How left and right brain hemispheres acquire neuroanatomical and cognitive specializations remains a mystery. The zebrafish is a powerful genetic model to explore the developmental basis of laterality in the vertebrate brain. The larval dorsal diencephalon consists of an asymmetric pineal complex and adjacent paired nuclei, the medial habenulae (Ha), which exhibit left-right differences in size, organization, neuropil density, and patterns of gene expression. In all vertebrates, axons from the medial habenulae project within a prominent fiber bundle to a shared midbrain target, the interpeduncular nucleus (IPN), serving as an important relay between the basal forebrain and brainstem nuclei. In zebrafish, habenular projections from the left and right sides of the brain innervate the target differently, in part due to a molecular asymmetry in an axon guidance cue. Analysis of the zebrafish Ha-IPN conduction system has led to a model whereby a slight anatomical asymmetry in one part of the brain, the pineal complex, can influence an adjacent region and its connections, triggering a cascade of differences throughout the brain. This model has implications for the study of many developmental neurological disorders, including schizophrenia, which had been previously linked to abnormalities in brain laterality. The overall goal of the proposed study is to characterize the zebrafish Ha-IPN system in greater detail using a unique set of asymmetrically expressed and region specific molecular markers. Subnuclear regions of the habenular nuclei will be carefully defined and new transgenic tools applied to trace their distinct afferent and efferent projections. A mutation recently identified as mapping to the zebrafish wntless gene, that affects development of the Ha-IPN system, will be analyzed to test the hypotheses that Wnt signaling influences the proliferation of habenular precursor cells and the establishment of brain asymmetry. An ongoing mutagenesis screen will identify new mutations that perturb the development and asymmetry of the dorsal diencephalon, or are essential for the formation of Ha-IPN connectivity. These fundamental studies in a vertebrate genetic model will provide much needed insight into poorly understand yet evolutionarily conserved brain regions, which mediate diverse behaviors and have been implicated in neurodevelopmental disorders. PUBLIC HEALTH RELEVANCE: Specialization of the left and right hemispheres is essential for normal brain function and abnormalities in brain laterality have been linked to a number of developmental neurological conditions, including schizophrenia. The proposed experiments using a genetic model system, the zebrafish, will increase our knowledge of how left-right differences arise in the developing brain by characterizing asymmetry in a highly conserved yet poorly understood forebrain to midbrain neural conduction pathway.